Novel Inositol Phospholipid Headgroup Surrogate Crystallized in the Pleckstrin Homology Domain of Protein Kinase B

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1 Novel Inositol Phospholipid Headgroup Surrogate Crystallized in the Pleckstrin Homology Domain of Protein Kinase B Stephen J. Mills,, David Komander,,, Melanie N. Trusselle, Stephen T. Safrany, Daan M. F. van Aalten, and Barry V. L. Potter, * Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, BA 7AY, U.K., Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, DD 5EH, U.K., and Department of Pharmacy and Pharmacology, University of Bath, BA 7AY, U.K., Present address: Institute of Cancer Research, Chester Beatty Laboratories, 37 Fulham Road, London SW3 6JB, U.K., These authors contributed equally to this work. ABSTRACT Protein kinase B (PKB/Akt) plays a key role in cell signaling. The PH domain of PKB binds phosphatidylinositol 3,4,5-trisphosphate translocating PKB to the plasma membrane for activation by 3-phosphoinositide-dependent protein kinase. The crystal structure of the headgroup inositol,3,4,5-tetrakisphosphate PKB complex facilitates in silico ligand design. The novel achiral analogue benzene,,3,4-tetrakisphosphate (Bz(,,3,4)P 4 ) possesses phosphate regiochemistry different from that of and surprisingly binds with similar affinity as the natural headgroup. Bz(,,3,4)P 4 co-crystallizes with the PKB PH domain in a fashion also predictable in silico. The -phosphate of Bz(,,3,4)P 4 does not interact with any residue, and the D5-phosphate of is not mimicked by Bz(,,3,4)P 4. Bz(,,3,4)P 4 is an example of a simple inositol phosphate surrogate crystallized in a protein, and this approach could be applied to design modulators of inositol polyphosphate binding proteins. *Corresponding author, b.v.l.potter@bath.ac.uk. Received for review January 9, 007 and accepted March 9, 007. Published online April 3, /cb70009r CCC: $ by American Chemical Society The protein kinase B (PKB, also known as Akt) subfamily of protein kinases consists of three members (PKB / Akt, PKB /Akt, and PKB /Akt3) with distinct cellular functions ( 3). The activation mechanism of PKB has been extensively studied (4) and depends on the generation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) from phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P ) on the plasma membrane by phosphoinositide 3-kinase (5). The pleckstrin homology (PH) domain of PKB binds to PtdIns(3,4,5)P 3, and this interaction recruits PKB to the plasma membrane (6); PKB is subsequently activated by phosphorylation at two sites (7). The PH domain of PKB can also bind phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P ) (8 0), and the headgroup derivatives myo-inositol,3,4,5-tetrakisphosphate (, a and b in Figure ) and myo-inositol,3,4-trisphosphate (Ins(,3,4)P 3 ). Importantly, the 5- phosphate of both PtdIns(3,4,5)P 3 and is solvent-exposed and not actively contributing to PKB PH domain binding (). Cellular levels of PtdIns(3, 4,5)P 3 are tightly controlled, and PKB activation is negatively regulated by the tumor suppressor phosphatase PTEN (protein phosphatase and tensin homologue deleted on chromosome 0), which dephosphorylates PtdIns(3,4,5)P 3 at the D3- position (). In cells lacking PTEN, PKB is constitutively active, which exerts its antiapoptotic effects and leads to tumor growth. Therefore, specific PKB inhibitors may deliver a targeted strategy to address patients who suffer from PTEN-induced tumors (3). Crystal structures of bound to the PH domains of a number of proteins have been reported; two of these are PKB () and 3-phosphoinositide-dependent protein kinase (PDK-) (4). The design of ligands that interact with PH domains is attractive, potentially providing tools that decouple PtdIns binding from kinase activation. There is a recognized but unmet need to develop simple mimics of inositol polyphosphates (5). Benzene polyphosphates are such molecules, in which the inositol ring is replaced by an aromatic core (6), but their application has never been evaluated in a structural context. nly the phosphates can engage in any flexible movement for benzene polyphosphates; additionally, even this will be different compared to an inositol polyphosphate, and any conformational flexibility derived from the inositol ring will be blocked. We discovered that benzene,,3,4- tetrakisphosphate (Bz(,,3,4)P 4, ) binds to and co-crystallizes with the PH domain of PKB. Bz(,,3,4)P 4 is one of three regioisomeric benzene tetrakisphosphates and wassynthesized from,3,4-trihydroxybenzalde- 4 ACS CHEMICAL BILGY VL. N.4

2 H H CH H Bz(,,3,4)P 4 H P P 4 3 P 5 H a (i) Cs C 3, DMF, BnBr, 80 C, h, 9% Bn P CH Bn Bn Bn Bn Bn (vi) TMSBr, CH Cl (5 ml), days, then MeH (5 ml); purification over Q-Sepharose Fast Flow using 0.0 M TEAB, 69% hyde (3 in Figure ). Triol 3 was benzylated to afford,3,4-tri--benzyloxybenzaldehyde 4, which was oxidized to the formate ester 5 and then hydrolyzed to give,3,4-tribenzyloxyphenol 6. The benzyl groups were removed to give,,3,4-tetrahydroxybenzene 7 (7), and the hydroxyl groups were phosphorylated using a modified method initially developed by Silverberg (8) to give compound 8 in 60% yield. The benzyl protecting groups of 8 were deblocked to give the crude product, which was purified over a column of Q-Sepharose Fast Flow using a gradient of triethylammonium bicarbonate buffer to give pure tetrakisphosphate in 69% yield. Importantly, using quantitative timeresolved FRET (TR-FRET) analysis of the glutathione S-transferase (GST)-tagged PH domain in complex with Eu-chelated anti- GST antibody, biotinylated PtdIns(3,4)P, and streptavidin-coated allophycocyanin (APC) (4), it was found that the pic 50 for Bz(,,3,4)P 4 binding to PKB is (mean standard error of the mean (Bn) P 8 H H P P P 3 P 4 P b Bz(,,3,4)P 4 P (ii) 3-Chloroperoxybenzoic acid CH Cl (Bn) P Bn P(Bn) P(Bn) CH (iii) Conc. HCl-H -MeH (:5:00), 84% Bn (SEM), n 4), giving a K i of 0.08 M (Supplementary Figure ). For comparison, the natural headgroup has a pic 50 of (mean SEM, n 5) and K i value of 0. M. The pic 50 for dic 8 - PtdIns(3,4,5)P 3 is (mean SEM, n 3), and K i is 0.3 M. Since Bz(,,3,4)P 4 has good affinity for the PH domain of PKB, it effectively acts as an inositol polyphosphate surrogate. Crystals of the PKB PH domain Bz(,,3,4)P 4 complex were grown after mixing purified PKB PH domain () with 0- fold molar excess of Bz(,,3,4)P 4. Highresolution (.94 Å) diffraction data were collected, and the structure was solved by molecular replacement using the PKB PH domain complex () as a search model. Unambiguous F o F c electron density for all atoms of the ligand was obtained (Figure ; Supplementary Table ). The overall structure of the PKB PH domain is not significantly different from the previously published structure (C root mean square deviation (rmsd) 0.55 Å). P P H 7 H Bn (iv) EtH, Pd(H), H, 86% (v) CH 3 CN, 30 C, CCl 4, N,N-diisopropylethylamine, H N,N-dimethylaminopyridine, H (Bn) P()H, 60% Figure. D-myo-Inositol,3,4,5-tetrakisphosphate (a and b) showing the inositol ring pucker and a D representation compared to Bz(,,3,4)P 4. H However, significant changes are observed in the binding mode of Bz(,,3,4)P 4 compared to (Figure 3, panel a; Supplementary Figure ). Both ligands occupy the same binding site on the PH domain, and the -, 3-, and 4-phosphates of Bz(,,3,4)P 4 form a hydrogen bonding pattern to the protein similar to that of the D-, D3-, and D4-phosphates of (Supplementary Table ). The /D-phosphate is closely enveloped by the Variable Loop- region (, residues 6 0), forming similar but not equivalent hydrogen bonds to backbone amide groups of the protein (Supplementary Table ). The fit atoms alignment tool was used within Sybyl 7.0 to determine the maximum possible overlap of Bz(,,3,4)P 4 and. The ligands were extracted from the experimental structures, and the equivalent heavy 4 3 Figure. Binding of Bz(,,3,4)P 4 to the PKB PH domain in the crystal structure. Unambiguous F o F c electron density (magenta), contoured at.5, is observed, covering all atoms of the ligand. Protein and ligand atoms are colored with purple phosphorus atoms, red oxygen atoms, and blue nitrogen atoms. Hydrogen bonds are indicated as black dotted lines. VL. N

3 a b Bz(,,3,4)P 4 Tyr8 Figure 3. Superposition of crystallographic and modeled ligands at the PKB PH domain. a) Comparison of the crystal structures reveals that the respective phosphorus atoms of the phosphate groups of (line) and Bz(,,3,4)P 4 (stick) involved in the protein interaction are located within. Å (/D-phosphate and 3/D3-phosphate) and 0.3 Å (4/D4-phosphate) of each other. Interestingly, the PKB PH domain does not bind to phosphoinositides lacking the D4-phosphate; furthermore, mutation of or, both of which contact the D4-phosphate of, block PKB PH domain ligand binding (). The observation that the 4-phosphate of Bz(,,3,4)P 4 is superposing perfectly with the D4-phosphate of, indicates the relative importance of this phosphate binding site. b) An overlay of the crystal structure Bz(,,3,4)P 4 (brown) and docked Bz(,,3,4)P 4 using GLD (colored by atom types) bound at the PH domain of PKB. atoms were aligned using a least squares fit. The lowest rmsd of equivalent atoms (retaining the,3,4-trisphosphate regiochemistry) is.04 Å, and the aromatic ring remains tilted with respect to the cyclohexane ring (33. between the planes created by the two rings). However, when the PH domains are superposed the rmsd between equivalent atoms of and Bz(,,3,4)P 4 is comparatively high (6.09 Å over heavy atoms only). The -, 3-, and 4-phosphates are below the plane of the ring and interact with PKB via hydrogen bonds, while the -phosphate is above the plane of the ring and does not engage in H-bonding (Supplementary Figure ). Low energy conformations of Bz(,,3,4)P 4 (see Supplementary Methods) reveal that the - and the 3-phosphates are restricted to opposite faces of the aromatic ring. Modeling was used to investigate whether the crystallographic binding modes could be reproduced. and Bz(,,3,4)P 4 were docked into PKB to assess if the GLD docking program could predict the crystallographic binding modes of both compounds. The two orientations (crystal structure and docked) were overlaid and their accuracy evaluated (Figure 3, panel b; Supplementary Figure 3). These structures were a good match (rmsd.4 Å over equivalent heavy atoms), and the docked model could be used for evaluating other potential binding partners and more drug-like molecules. Stereoview diagrams of,3,4-trisphosphate regiochemistry for recognition by PKB PH domain is provided by Bz(,,3,4)P 4, with for comparison (Figure 4, panels a and b). PKB inhibitors are a priority target of the pharmaceutical industry. Most inhibitors target the ATP binding site of the kinase domain and often prove to be non-specific (9, 0). Therefore, small molecules that specifically target the PH domain of PKB should block its membrane localization and hence the conformational change prior to PKB activation (3). The charges of benzene polyphosphates could be masked with acetoxymethyl or pivaloyloxymethyl groups to provide cell-permeable derivatives, enhancing their value as potential modulatory tools for cell biology. The addition of to fulllength PKB in an in vitro kinase assay is insufficient to induce PKB activation (), and whether PKB binds to soluble inositol phosphates in vivo is debated (). Bz(,,3,4)P 4 has good affinity for the PKB PH domain, as determined by TR-FRET. Recent data () show that could not compete with PtdIns(3,4,5)P 3 binding at the PH domain of PKB, whereas other data show an affinity of 68 M (3). Affinities of.5 M and higher have also been reported (9, 4). There is no explanation for these differences at present; however, inositol phosphates clearly play an important role in regulating proteins with PH domains, for example, phospholipase C (PLC) (5) and PDK- localization in vivo (4). We describe the crystal structure of the novel PtdIns(3,4,5)P 3 headgroup mimic Bz(,,3,4)P 4 in complex with the PH domain of PKB (Figure ; Supplementary Table ). ur modeling approaches indicate that the PKB-PH domain provides a robust scaffold for in silico ligand design. This is crucial, since more lipid-like derivatives that might directly interfere with PKB activation (similar to PtdIns(3,4,5)P 3 ether analogues) may be unsuitable for co-crystallization studies. We also demonstrate that for Bz(,,3,4)P 4 only the three phosphate groups equivalent to the,3,4-trisphosphate are required for binding, correlating with other crystallographic observations. There are many proteins that bind inositol phosphates and phospholipids. Enzymeresistant benzene polyphosphates could be used to replace inositol polyphosphates in other crystallization experiments where inositol polyphosphates may be modified by proteins such as phosphatases or kinases. Such inositol phosphate surrogates could be structurally tailored for each protein and should provide new tools to inhibit various inositol polyphosphate binding proteins and phosphoinositide modifying enzymes, as we have recently reported (6, 7). METHDS Chemistry. Experimental details are similar to those described previously (6). Crystallography of PKB PH Domain in Complex with Bz(,,3,4)P 4. The PKB PH domain protein was expressed and purified as described previously (). The triethylammonium salt of Bz(,, 3,4)P 4 was solubilized directly in an 8.5 mg/ml PKB PH domain solution to achieve a 0-fold molar excess of ligand over protein and incubated on ice for h. Crystals of the protein ligand complex were grown using the hanging drop vapor diffusion method derived from several conditions similar to those reported for the PKB PH domain 44 VL. N MILLS ET AL.

4 a b D3 3 4 complex (5 30% poly-(ethylene glycol) 3000, 0. M sodium acetate trihydrate (ph ), 0. M ammonium acetate) (). Crystals were grown overnight at 0 C and reached their maximum size after 5 d. Prior to freezing, the crystals were soaked in crystallization solution enriched with 0% -methyl-,4- pentanediol. Diffraction data to a resolution of.94 Å were collected at the European Synchrotron Radiation Facility in Grenoble, station ID4, at 00 K, and the structure was solved by rigid body refinement using CNS (8). Subsequently, refinement was carried out in CNS, including extensive simulated annealing to reduce phase bias. Ligand topologies were generated using PRDRG (9). See Supplementary Table for data collection and refinement statistics. FRET Binding Experiments. The GST-tagged PH domain of PKB was prepared as described in Supplementary Methods. The quantitative TR-FRET analysis of binding was performed in white 96-well D D D4 D5 D3 3 4 D Figure 4. Hydrogen bonding of PKB PH domain to InsP 4 and BzP 4. a) Stereoview of Bz(,,3,4)P 4 bound at the PH domain of PKB. b) Stereoview of bound at the PH domain of PKB. Protein and ligand atoms are colored with purple phosphorus atoms, red oxygen atoms, and blue nitrogen atoms. Hydrogen bonds are indicated as dotted lines. D D4 D5 plates (Greiner) using a BMG Labtech PHERAstar with the following settings: excitation 337 nm filter, emission 665 and 60 nm filters, 300 flashes per well, 0 s flashes, read for 400 s following a 50 s delay. Assays were performed in a buffer made of HEPES, 50 mm, ph 6.8; NaCl, 50 mm; MgCl, 5 mm; DTT, 5 mm; CHAPS, 0.5%; EDTA, mm in a final assay volume of 50 L. The K d values for biotinylated dic 8 -PtdIns(3, 4)P were determined by increasing amounts (0 00 nm) of biotinylated dic 8 -PtdIns(3,4)P (Cell Signals), which were incubated with GSTtagged PH domain of PKB (0 nm) in the presence of excess ( nm) europium-labeled goat anti-gst antibody (Perkin Elmer) and streptavidinconjugated APC. The binding of the biotinylated lipid to the protein allows FRET to occur between europium (donor) and APC (acceptor). Fluorescence was monitored at 665 and 60 nm; the ratio of these signals allows the determination of relative amount of binding (Supplementary Figure ). The pk d for biotinylated dic 8 - PtdIns(3,4)P is (mean SEM, n 3), and K d of 5.9 nm. Competition assays were performed with a fixed amount (5 nm) of biotinylated dic 8 -PtdIns(3,4)P and increasing amounts of Bz(,,3,4)P 4, with as a control (Supplementary Figure ). IC 50 values were determined, and K i values were calculated (30). All curve fitting and statistical analysis was performed using Prism (GraphPad). Synthesis of,3,4-tribenzyloxybenzaldehyde (4).,3,4-Trihydroxybenzaldehyde 3 (3.08 g, 0 mmol), cesium carbonate (3.58 g, 00 mmol), and benzyl bromide (.9 ml, 00 mmol) in dry DMF (00 ml) were stirred for h at 80 C. The yellow-brown solution was filtered over a bed of Celite and washed with acetone (00 ml). The solvents were evaporated, giving a red-brown oil that was partitioned between CH Cl and water (00 ml of each). The organic layer was dried, and the solvent was evaporated to give the crude compound. Compound 4 was purified by flash chromatography (CH Cl ) to give a white solid, R f 0.40 (CH Cl ), 7.73 g (9%), mp C (hexane). Synthesis of,3,4-tribenzyloxyphenol (6). 3-Chloroperoxybenzoic acid (.9 g, 6.8 mmol) was added to a solution of,3,4-tri--benzyloxybenzaldehyde 4 (4.45 g, 0 mmol) in dry CH Cl (00 ml), and the mixture was stirred for 0 h at RT. The organic layer was washed with an aqueous solution of 0% sodium metabisulfite, a saturated solution of sodium hydrogen carbonate ( 00 ml of each), and water (00 ml). The organic layer was dried, and the solvent was evaporated to give the crude formate ester 5 as a yellow syrup, R f 0.56 (CH Cl ). Compound 5 was dissolved in methanol (50 ml) containing 0.5 ml of concentrated HCl and.5 ml of water and then stirred for h at RT. The reaction was neutralized using solid NaHC 3 (5 g), the remaining solid was filtered, and the solvent was evaporated. Compound 6 was purified by flash chromatography (CHCl 3 ) and isolated as a pale yellow oil, R f 0.40 (CHCl 3 ), g (84%). Synthesis of,,3,4-tetrahydroxybenzene (7).,3,4-Tribenzyloxyphenol 6 (3.36 g, 8.5 mmol) was dissolved in ethanol (00 ml) followed by the addition of 0% palladium hydroxide (500 mg). The air was expelled from the vessel, and the mixture was stirred over an atmosphere of hydrogen for 4 h at RT. The solution was filtered over a bed of Celite, and the solvent was evaporated giving a beige solid, R f 0.40 (CH Cl /MeH 9:), 993 mg (86%); mp 6 C (ether/hexane). Synthesis of,,3,4-tetrakis(dibenzyloxyphosphoryloxy)benzene (8). Carbon tetrachloride (.93 ml, 0 mmol), N,N-diisopropylethylamine (.46 ml, 8.4 mmol), a catalytic amount of N,Ndimethylaminopyridine (49 mg, 0.40 mmol), and dibenzylphosphite (.33 ml, 6 mmol) were stirred in dry acetonitrile (30 ml) and cooled using dry ice alone.,,3,4-tetrahydroxybenzene 7 (4 mg,.0 mmol) was added to the solution over 5 min. The solution was stirred for a further h, allowing the reaction to warm to RT. The organic solvent was evaporated, dichloromethane (00 ml) was added and washed with water (00 ml), and VL. N

5 the organic solvent was evaporated. Compound 8 was purified by flash chromatography using ethyl acetate/hexane (:) to provide 706 mg (60%) as a colorless syrup, R f 0.0 (ether), R f 0.46 (Et- Ac/hexane :). Synthesis of Benzene,,3,4-tetrakisphosphate (). Compound 8 (9 mg, 00 mol) and bromotrimethylsilane (.0 ml, 7.58 mmol) were stirred in dry CH Cl (5 ml) for d under an atmosphere of nitrogen. The solvents were evaporated to give a syrup that was dissolved in methanol (5 ml) and then stirred for 5 min. Compound was purified by ion exchange chromatography over Q-Sepharose Fast Flow using a gradient of TEAB (0.0 M) and eluted between. and.8 M buffer to give a glassy triethylammonium salt, 69 mg (69 mol, 69%). PyML was used to generate all figures. Accession Codes: The coordinates of the PKB PH domain Bz(,,3,4)P 4 complex have been submitted to the Protein Data Bank and are listed as UVM. Acknowledgment: We thank the Wellcome Trust for Programme Grant support ( to B.V.L.P) and acknowledge useful discussions with A. M. Riley. Torsten Werner is acknowledged for help in protein production and crystallization. D.K. was supported by a Medical Research Council Predoctoral Fellowship and is now a Beit Memorial Fellow. S.T.S. is a Royal Society University Research Fellow who thanks the Royal Society for financial support. We thank BMG LABTECH for use of their PHERAstar plate reader and G. D. Holman and F. Koumanov (University of Bath) for assistance with TR-FRET assays. Supporting Information Available: This material is available free of charge via the Internet. REFERENCES. Lawlor, M. A., and Alessi, D. R. (00) PKB/Akt: a key mediator of cell proliferation, survival and insulin responses? J. Cell Sci. 4, Fayard, E., Tintignac, L. A., Baudry, A., and Hemmings, B. A. (005) Protein kinase B/Akt at a glance, J. Cell Sci. 8, Bellacosa, A., Testa, J. R., Moore, R., and Larue, L. 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(00) The PyML Molecular Graphics System, DeLano Scientific, San Carlos, CA. 46 VL. N MILLS ET AL.